Bottom Line:
However, IPS-1 is essential for both BMDC activation and IFN production.In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection.However, only RIG-I-/- cells exhibit a delay in type I IFN production.

ABSTRACTAs with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5-/- and RIG-I-/- mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I-/- cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1-/- mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.

ppat-1001016-g006: RABV infection triggers IFN production and DC activation via either Mda-5 or RIG-I.CD86 expression on BMDC following RABV infection was analyzed by flow cytometry 12, 24, and 48 h post infection. (A–B) For each sample the fold increase in activation was determined by dividing the geometric mean fluorescent intensity (MFI) of the sample by the MFI of the uninfected sample for Mda-5−/− (A) and RIG-I−/− (B). Each point in A and B is representative of BMDCs from one mouse and data for RIG-I is representative of two independent experiments. (C) IFN-α/ß levels were monitored by infectivity of VSV-GFP on reporter cells. Reporter cells were pre-treated with UV-inactivated BMDC supernatant from RABV infection. 24h after supernatant was applied, reporter cells were infected with VSV-GFP. Fluorescence indicates viral replication, and thus, lack of type I IFN. Statistical significance was determined by T-test analysis and any significance is indicated, *p<0.05, **p<0.01.

Mentions:
Mda-5 mediated induction of IFN-ß has been described to occur in response to plus-stranded RNA viruses like picornaviruses, whereas it is reported that RIG-I is responsible for type I IFN induction in response to rhabdovirus infection [34]. However, the function of Mda-5 in the innate immune response to rhabdoviridae has not yet been elucidated. Furthermore, the role of these PRRs following a RABV infection in DCs remains unknown. Therefore we wanted to determine which of the two receptors recognizes RABV. For this approach, BMDCs from Mda-5−/− mice and RIG-I−/− mice were isolated. As shown in Figure 6A, Mda-5−/− BMDCs express high levels of CD86 on their surface at 24 and 48 hpi. Of note, there is a significant reduction of CD86 surface expression on Mda-5−/− BMDCs at 12 hpi when compared to wildtype cells. Likewise, RIG-I −/− BMDCs also have a defect in BMDC activation at 12 hpi, while CD86 expression at 24 and 48 hpi is equal for RIG-I−/− and RIG-I+/+ cells (Figure 6B). In addition, it appears that while Mda-5−/− cells are able to induce type I IFN expression 12 hpi, RIG-I−/− cells have an early defect in type I IFN induction. Importantly, by 48hpi, RIG-I−/− BMDC do produce enough type I IFN to suppress VSV-GFP replication (Figure 6C). This indicates that RABV can induce BMDC activation and type I IFN via both Mda-5 and RIG-I ligation. Furthermore, any perturbation in IPS-1 mediated signaling cascades seems to affect the early response (12hpi) to RABV.

ppat-1001016-g006: RABV infection triggers IFN production and DC activation via either Mda-5 or RIG-I.CD86 expression on BMDC following RABV infection was analyzed by flow cytometry 12, 24, and 48 h post infection. (A–B) For each sample the fold increase in activation was determined by dividing the geometric mean fluorescent intensity (MFI) of the sample by the MFI of the uninfected sample for Mda-5−/− (A) and RIG-I−/− (B). Each point in A and B is representative of BMDCs from one mouse and data for RIG-I is representative of two independent experiments. (C) IFN-α/ß levels were monitored by infectivity of VSV-GFP on reporter cells. Reporter cells were pre-treated with UV-inactivated BMDC supernatant from RABV infection. 24h after supernatant was applied, reporter cells were infected with VSV-GFP. Fluorescence indicates viral replication, and thus, lack of type I IFN. Statistical significance was determined by T-test analysis and any significance is indicated, *p<0.05, **p<0.01.

Mentions:
Mda-5 mediated induction of IFN-ß has been described to occur in response to plus-stranded RNA viruses like picornaviruses, whereas it is reported that RIG-I is responsible for type I IFN induction in response to rhabdovirus infection [34]. However, the function of Mda-5 in the innate immune response to rhabdoviridae has not yet been elucidated. Furthermore, the role of these PRRs following a RABV infection in DCs remains unknown. Therefore we wanted to determine which of the two receptors recognizes RABV. For this approach, BMDCs from Mda-5−/− mice and RIG-I−/− mice were isolated. As shown in Figure 6A, Mda-5−/− BMDCs express high levels of CD86 on their surface at 24 and 48 hpi. Of note, there is a significant reduction of CD86 surface expression on Mda-5−/− BMDCs at 12 hpi when compared to wildtype cells. Likewise, RIG-I −/− BMDCs also have a defect in BMDC activation at 12 hpi, while CD86 expression at 24 and 48 hpi is equal for RIG-I−/− and RIG-I+/+ cells (Figure 6B). In addition, it appears that while Mda-5−/− cells are able to induce type I IFN expression 12 hpi, RIG-I−/− cells have an early defect in type I IFN induction. Importantly, by 48hpi, RIG-I−/− BMDC do produce enough type I IFN to suppress VSV-GFP replication (Figure 6C). This indicates that RABV can induce BMDC activation and type I IFN via both Mda-5 and RIG-I ligation. Furthermore, any perturbation in IPS-1 mediated signaling cascades seems to affect the early response (12hpi) to RABV.

Bottom Line:
However, IPS-1 is essential for both BMDC activation and IFN production.In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection.However, only RIG-I-/- cells exhibit a delay in type I IFN production.

ABSTRACTAs with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5-/- and RIG-I-/- mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I-/- cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1-/- mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.